This invention relates to novel water-soluble polypeptides having morphogenesis-accelerating activity and cell-proliferating activity against epithelial cells.
It is suggested that factors derived from mesenchyme cells existing around an epithelial tissue control the normal morphogenesis of the epithelial tissue. It is also recognized that many of the causes of diseases resulting from the abnormal morphogenesis of epithelial tissues are due to the aberration of mesenchyme cells. Based on such findings, there is interest in the elucidation of control mechanism of the morphogenesis of the epithelial tissue by the mesenchyme cells. However, since a group of substances participating in the control of morphogenesis of epithelial tissues by mesenchyme cells is expressed in a complex system under time and spatial restrictions, great difficulty exists in the isolation of these substances and the analysis of their functions. It is also difficult to construct a model experimental system that simplifies the morphogenesis of epithelial tissues. For these reasons and others, there has not been major progress in the studies of this field to date. Accordingly, it is earnestly desired that the control mechanism of morphogenesis of epithelial tissues be analyzed in order to elucidate the mechanism of crisis of diseases resulting from the morphogenesis of the epithelial tissues or to establish methods for the treatment of these diseases.
Under these circumstances epimorphin, which participates in the control of morphogenesis of epithelial tissues, was separated and purified. (Japanese Unexamined Application Publn. Hei 6-25,295.) It was shown that this substance is a physiologically active substance the core protein of which is a protein comprising from 277 to 289 amino acids and which is mainly biosynthesized by mesenchyme cells. It was also shown that epimorphin possesses an activity for accelerating the morphogenesis of epithelial tissues by acting the epithelial cells and that the normal morphogenesis does not take place under conditions where epimorphin does not exert its functions.
In addition, with respect to the structural characteristics of epimorphin, it has been found that structurally the epimorphin molecule is largely divided into four fragments. (European Patent Application Publn. No. 0698666.) Specifically, the polypeptide constituting the full-length epimorphin can be divided into, from its N-terminal side, a coiled coil domain (1), a functional domain (2), a coiled coil domain (3), and a hydrophobic domain at its C-terminal. As for the functional domain among these fragments (the domain defined by the 104th to 187th amino acids from its N-terminal in human epimorphin), it has been suggested that this domain participates in cell adhesion and is closely involved in manifestation of the biological activities of epimorphin. (European Patent Application Publn. No. 0698666 vide supra.)
Since epimorphin possesses an activity for accelerating the normal morphogenesis, it is expected that this substance is useful as a medicament for the prevention or treatment of diseases resulting from the aberration of morphogenesis, or as the active ingredient of medicaments such as a hair growth-promoting agent. However, native epimorphins obtained from mammals find difficulty in being put to practical use as medicaments, because they are sparingly soluble in aqueous media such as physiological saline solution. For this reason, attempts are made to de novo synthesize epimorphin derivatives that are excellent in water-solubility while substantially maintaining the morphogenesis-accelerating activity of the native epimorphins. For example, there is known an altered form (Fragment 123) that is obtained by deletion of the hydrophobic domain at the C-terminal. (Japanese Unexamined Application Publn. Hei 6-25,295.)
With respect to the coiled coil domain (1) which is a partial structure of epimorphin, it has previously been shown that this domain possesses an activity of endowing epimorphin with its solubility. However, it has also been shown that if part of the coiled coil domain (1) is removed by deletion of amino acids from the N-terminal of epimorphin, the cell adhesion activity of the resulting altered form has diminished. (European Patent Application Publn. No. 0698666.) More specifically, it has been disclosed that with regard to the activity of this domain, it positively contributes to the aspect of solubility by such activity as that altering a higher structure of Fragment 23 but that it negatively contributes to the cell adhesion activity by such activity as that masking the functional domain (2); and there has been a negative suggestion concerning the applicability in medicaments. Further, while there have been reports of the cell adhesion activity of each domain of the epimorphin (the coiled coil domain (1), the functional domain (2), or the coiled coil domain (3)) with respect to its physiological activity, the morphogenesis-accelerating activity similar to that of epimorphin has not been known thus far.
An object of this invention is to provide a water-soluble polypeptide having morphogenesis-accelerating activity against epithelial cells. More specifically, it is the object of the invention that a polypeptide be provided which acts on the epithelial cells to accelerate the morphogenesis of epithelial tissues and which is soluble in an aqueous medium such as physiological saline solution.
Another object of this invention is to provide a medicament that contains a polypeptide having the above-mentioned characteristics as an effective ingredient and that is useful for the prevention and/or treatment of a disease which results from of morphogenetic factors such as epimorphin or which involves the destruction of tissue or organ. A further object of the invention is to provide a hair growth-promoting agent containing the above-mentioned water-soluble polypeptide as an effective ingredient.
The present inventors made thorough efforts to attain the above-mentioned objects, and as a result, discovered that polypeptides comprising the coiled coil domain (1), which constitutes epimorphin, are soluble in aqueous media, act on epithelial cells to accelerate the morphogenesis of epithelial tissues, and possess marked cell propagation-accelerating activity against the epithelial cells. This invention has been accomplished based on the above-mentioned findings. As used herein, xe2x80x9ccell propagationxe2x80x94accelerating activityxe2x80x9d means that when cells are cultured in a serum-free medium for several days, it can increase the number of the viable cells. Namely, this invention provides a polypeptide having morphogenesis-accelerating activity, said polypeptide defined by the amino acid sequence (I) as described below (SEQ ID No. 1 in the Sequence Listing) which may, in certain cases, be referred to as xe2x80x9cpolypeptide (I)xe2x80x9d in the present specification:
Met Arg Asp Arg Leu Pro Asp Leu Thr Ala Cys Arg Lys Asn Asp Asp Gly Asp Thr Val Val Val Val Glu Lys Asp His Phe Met Asp Asp Phe Phe His Gln Val Glu Glu Ile Arg Asn Ser Ile Asp Lys Ile Thr Gln Tyr Val Glu Glu Val Lys Lys Asn His Ser Ile Ile Leu Ser Ala Pro Asn Pro Glu Gly Lys Ile Lys Glu Glu Leu Glu Asp Leu Asn Lys Glu Ile Lys Lys Thr Ala Asn Lys Ile Arg Ala Lys Leu Lys Ala Ile Glu Gln Ser Phe Asp Gln Asp Glu.
Also, the invention provides a polypeptide having morphogenesis-accelerating activity, said polypeptide defined by the amino acid sequence (II) as described below (SEQ ID No. 2 in the Sequence Listing) which may, in certain cases, be referred to as xe2x80x9cpolypeptide (II)xe2x80x9d in the present specification:
Met Arg Asp Arg Leu Pro Asp Leu Thr Ala Cys Arg Thr Asn Asp Asp Gly Asp Thr Ala Val Val Ile Val Glu Lys Asp His Phe Met Asp Gly Phe Phe His Gln Val Glu Glu Ile Arg Ser Ser Ile Ala Arg Ile Ala Gln His Val Glu Asp Val Lys Lys Asn His Ser Ile Ile Leu Ser Ala Pro Asn Pro Glu Gly Lys Ile Lys Glu Glu Leu Glu Asp Leu Asn Lys Glu Ile Lys Lys Thr Ala Asn Arg Ile Arg Gly Lys Leu Lys Ser Ile Glu Gln Ser Cys Asp Gln Asp Glu.
Further, the invention also provides the following: a polypeptide characterized by the substitution, addition, and/or deletion of one or more amino acids in the amino acid sequence (I), or the amino acid sequence (II); each above-mentioned polypeptide having substantially the same morphogenesis-accelerating activity as native epimorphins; and each above-mentioned polypeptide further having cell propagation-accelerating activity. As used herein, xe2x80x9cthe substitution, addition, and deletion of amino acid(s)xe2x80x9d can be carried out by the method that is referred to as xe2x80x9csite-directed mutagenesis,xe2x80x9d for example.
According to another embodiment of this invention, there is provided a medicament containing the above-mentioned polypeptide as an effective ingredient. As preferred embodiments of this medicament, there is provided a medicament for use in the prevention and/or treatment of a disease resulting from little expression morphogenetic factors or a disease involving the of destruction of tissue or organ, as well as a medicament for use as a hair growth-promoting agent. In addition to these, there are provided a method for the treatment and/or prevention of a disease resulting from little expression of morphogenetic factors or a disease involving the destruction of tissue or organ, which contains the step of administering the polypeptide to a mammal, including human; a method for diagnosing a disease resulting from little expression of epimorphin, which contains the step of administering the polypeptide to a mammal, including human; and a method for promoting the growth of hair, which contains the step of administering the polypeptide to a mammal, including human.
According to a further embodiment of this invention, there is provided an antibody, preferably a monoclonal antibody, that specifically recognizes the above-mentioned polypeptides. As preferred embodiments of the antibody, there is provided an antibody that specifically binds to the polypeptides and inhibits the morphogenesis-accelerating activity of the polypeptides against epithelial cells, as well as an antibody that specifically binds to an epimorphin having a coiled coil domains (1) and inhibits the morphogenesis-accelerating activity of the epimorphin against epithelial cells. There is also provided a medicament containing the antibody as an effective ingredient, preferably a medicament useful in the prevention and/or treatment of a disease resulting from the excessive expression of epimorphin, as well as a medicament for use as a hair growth-promoting agent. In addition to these, there are provided a method for the treatment and/or prevention of a disease resulting from the excessive expression of epimorphin, which contains the step of administering the antibody to a mammal, including human; a method for diagnosing a disease resulting from the excessive expression of epimorphin, which contains the step of administering the antibody to a mammal, including human; and a method for inhibiting the growth of hair, which contains the step of administering the antibody to a mammal, including human.